The present invention relates to a novel class of cyclophilins which contain a tyrosine residue as opposed to tryptophan or histidine in the drug binding site as well as to a method for identifying anti-parasitic compounds. More specifically, the present invention relates to a method for the identification of compounds capable of binding and/or inhibiting cyclophilins containing a tyrosine residue in lieu of tryptophan/histidine in the drug binding pocket, as well as to methods of treating parasitic infections which are not susceptible to cyclosporin A.
Cyclosporin A (CsA) is a lipophilic, 11 amino acid cyclic peptide originally isolated from the fungus Tolypocladium inflatum. Its immunosuppressive properties were first described in 1978 (Borel, Pharmacol. Rev. 41:259-371 (1990)) and it is currently the drug of choice in transplantation surgery and in the treatment of various autoimmune diseases (Kahan, xe2x80x9cCyclosporin: Biological activity and clinical applications,xe2x80x9d Grune and Stratton, Orlando, Fla. (1983)).
In 1984 the receptor for CsA was identified and purified from bovine spleen, and named cyclophilin A (CypA) (Handschumacher, et al., Science, 226:544-547 (1984)). CypA is an 18-kDa cytoplasmic protein (Haendler, et al., EMBO. J, 6:947-950 (1987)) that is abundantly expressed in all mammalian tissues (Koletsky, et al., J. Immunol. 137:1054-1059 (1986)). More recently, other cyclophilin isoforms have been described which share the highly conserved 18-kDa domain flanked by unique domains which are thought to function in organelle and membrane targeting of the protein (Gething, et al., Nature 355:33-45 (1992), Price, et al., PNAS, 88:1903-1907 (1991), Spik, et al., J. Biol. Chem. 266:10735-10738 (1991), Friedman, et al., Cell 66:23204-23214 (1991), and Bergsma, et al., J. Biol. Chem. 266:23204-231214 (1991)). These include from humans the larger Cyp-40 (40 kDa) (Kieffer, et al., J. Biol. Chem. 267:5503-5507 (1992)) and Cyp-60 (60 kDa) (Wang, et al., Biochem. J. 314:313-319 (1996)) proteins, and the surface-associated natural killer (NK) cell cyclophilin (150 kDa) (Anderson,.et al., PNAS, USA 90:542-546 (1993)).
Cyclophilins have also been found in several parasites including Schistosoma mansoni (Koletsky, et al., J. Immunol, supra, Klinkert, et al., Mol. Biochem. Parasitol., 75:99-111 (1995), Kiang, et al., Mol. Biochem. Parasitol., 76:73-82 (1995)), Echinococcus granulosus (Lightowlers, et al., Mol. Biochem. Parasitol., 36:287-289 (1989), Schistosoma japonicum (Argaet, et al., J. Parasitol., 78:660-664 (1992)), Toxoplasma gondii (High, et al., J. Biol. Chem., 269:9105-9112 (1994)), Plasmodium falciparum (Bell, et al., Biochem. Pharmacol., 48:495-503 (1994) and Reddy et al., Mol. Biochem. Parasitol., 73:111-121 (1995)), Hymenolepis microstoma (Roberts, et al., Parasitology, 111:591-597 (1995)), and the filarial worms Brugia malayi (Ma, et al., Mol. Biochem. Parasitol., 79:235-241 (1996) and Page, et al., Parasitol. Today, 11:385-388 (1995)), Onchocerca volvulus and Dirofilaria immitis (Ma, et al., Mol. Biochem., Parasitol. supra and Hong, et al., Exp. Parasitol., in press). Multiple isoforms can exist in parasites since 2 forms have been found in T. gondii (High, et al., J. Biol. Chem., supra) and filarial parasites (Ma, et al., Mol. Biochem. Parasitol, supra, Hong, et al., Exp. Parasitol, in press, supra, and Page, et al., Biochemistry, 34:11545-11550 (1995)).
In addition to binding CsA, CypA was subsequently shown to possess an enzymatic activity (Fischer, et al., Biomed. Biochim. Acta, 43:1101-1111 (1984)). Fischer and coworkers characterized a new enzyme from pig kidney which was capable of catalyzing the cis to trans interconversion of proline containing peptides, and hence named it peptidyl-prolyl cis-trans isomerase (PPlase). Subsequent N-terminal peptide sequencing of this enzyme revealed that it was identical to cyclophilin (Lang, et al., Nature, 329:268-270 (1987)).
PPlases catalyse the cis-trans isomerisation of proline-imidic peptide bonds in oligopeptides and accelerate the refolding of several proteins in vitro (Gething, et al., Nature, supra, Lang, et al., Nature, 329:268-270 (1987) and Fransson, et al., FEBS Lett., 296:90-94 (1992)) and in vivo (Lodish, et al., J. Biol. Chem., 266:14835-14838 (1991) and Steinmann, et al., J. Biol. Chem., 266:1299-1303 (1991)). PPlases also function as protein chaperones (Freskgard, et al., Science, 258:466-468 (1992) and Rinfret, et al., Biochemistry, 33:1668-1673 (1994)). These properties suggest that cyclophilins may also have a critical role in parasite development.
Every cyclophilin examined to date has PPlase activity, including the CypA homologs present in S. mansoni (Koletsky, et al, J. Immunol., supra),T. gondii (High, et al., J. Biol. Chem., supra) and P. falciparum (Bell, et al., Biochm. Pharmacol., supra). Recombinant B. malayi cyclophilins were also found to possess high levels of PPlase activity (Ma, et al., Mol. Biochem., Parasitol, supra and Page, et al., Biochemistry, supra)
In most cases, drug binding results in inhibition of PPlase activity (Takahashi et al., Nature, 337:473-475 (1989). X-ray crystallography (Pflugl, et al., Nature, 361:91-94 (1993) and site-directed mutagenesis studies (Liu, et al., Biochemistry 30:2305-2310 (1991)) have determined that 13 specific residues comprise the drug binding site of CypA, namely, Arg-Phe-Met-Gln-Gly-Ala-Asp-Gln-Gln-Phe-Trp-Leu-His (SEQ ID NO:16). These residues are highly conserved among most cyclophilin isoforms and homologs. Liu and coworkers demonstrated that the tryptophan residue at position 121 of CypA is particularly important for drug binding. The same 13 amino acids, notably including tryptophan, are found in the CsA-sensitive cyclophilins from E. granulosus (Lightowlers, et al., Mol Biochem. Parasitol, supra), T. gondii (Argaet, et al., J. Parasitol., 78:660-664 (1992), P falciparum (Bell, et al., Biochem., Pharmacol., supra, Reddy, Mol. Biochem. Parasitol., 73:111-121 (1995)), and the filarial Cyp-2 cyclophilins (Ma, et al., Mol. Biochem., Parasitol., supra). Cyclophilins which have a residue other than tryptophan in the critical position have been reported. Human Cyp-40 (kietten, et al., J. Biol. Chem., supra) and NK cell cyclophilin (Anderson, et al., PNAS, supra) have histidine, and human Cyp-60 (Wang, et al., Biochem. J., supra) has a tyrosine residue in this position. The Cyp-1 proteins from filarial parasites (Page, et al., Biochemistry, supra, Hong, et al., Exp. Parasitol., supra) and certain cyclophilins from C. elegans (Page, et al., Biochem. J., 317:179-185 (1996)) also have a histidine residue in the critical position. We have determined that this amino acid difference was shown to be responsible for the lack of sensitivity of the Cyp-1 PPlase activity to inhibition with CsA
Various cDNA libraries of B. malayi and O. volvulus are currently being analyzed through tag sequencing (EST) analysis and sequences deposited in GenBank (Blaxter, et al., Mol. Biochem. Parasitol., 77:77-93 (1996). Sequences related to both human Cyp-60 (xe2x80x98tyrosine-containingxe2x80x99 cyclophilin) and a PPlase from Schizosaccharomyces pombe (xe2x80x98histidine-containingxe2x80x99 cyclophilin) have been found in B. malayi (accession numbers W15136, AA111775) and O. volvulus (accession number AA294728). Based on these sequence deposits alone, there is insufficient information available to identify any of these sequences as belonging to xe2x80x98tyrosine-containingxe2x80x99 cyclophilins. In accordance with the present invention, it has been determined that these 3 partial sequences are related to DiCyp-3.
CsA has also been demonstrated to posses a broad spectrum anti-parasitic activity (Page, et al., Parasitol. Today, supra, and Chappell, et al., Parasitology, 105 Supplement, S25-S40 (1992)). The parasites S. mansoni (Bueding, et al., Agents Actions 11:380-383 (1981)), T. gondii (Mack, et al., Antimicrob Agents Chemother, 26:26-30 (1984) and McCabe, et al., Transplantation, 41:611-615 (1986)) and P. falciparum (Thommen-Scott, Agents Actions, 11:770-773 (1981)) are adversely affected by the drug and the PPlase activity of their cyclophilins is strongly inhibited by nanomolar concentrations of CsA (Koletsky, et al., J. Immunol., supra, High, et al., J. Biol. Chem., supra, Bell, et al., Biochem. Pharmacology, supra, Reddy, et al., Mol. Biochem. Parasitol., supra). More recently, a non-immunosuppressive derivative of CsA was demonstrated to have potent activity against malaria parasites (Bell, et al., Biochem. Pharmacol., supra). In contrast, Brugia malayi (Page, et al., Parasitol. Today, supra) is not susceptible to CsA, and previous studies have shown that this parasite possesses both a CsA-insensitive (Cyp-1) (Page, et al., Biochemistry, supra) and -sensitive (Cyp-2) cyclophilin (Ma, et al., Mol. Biochem., Parasitol., supra). Cyp-1 was demonstrated to have a histidine residue in the critical tryptophan position (Page, et al., Biochemistry, supra, U.S. Pat. No. 5,482,850). We have determined using site-directed mutagenesis studies that the histidine residue is responsible for drug insensitivity. Cyp-1 and Cyp-2 homologs have also been identified in O. volvulus and D. immitis (Ma, et al., Mol. Biochem., Parasitol, supra and Hong, et al., Exp. Parasitol., supra).
For tyrosine-containing cyclophilin (Cyp-3) described by the present inventors it would be desirable to have a compound that inhibits the PPlase activity of these proteins. Such a compound may be used to treat parasites which are not susceptible to the anti-parasitic effects of CsA
It would also be desirable to have a method which can be used to readily screen and select compounds that are capable of binding tyrosines-containing cyclophilins from parasites which are not susceptible to the anti-parasitic effects of CsA and/or which inhibit the PPlase activity of such proteins. More specifically, it would be desirable to have a method which can be used to screen and select CsA derivatives that are capable of binding such cyclophilins and inhibiting PPlase activity while having reduced immunosuppressive activity on the host.
In accordance with the present invention, it has been discovered that parasites which are not susceptible to the anti-parasitic effects of CsA possess cyclophilins in which the conserved tryptophan at the CsA binding domain has been substituted with another amino acid, in particular substituted with tyrosine. The present invention relates to these novel cyclophilins as well as to the use of these xe2x80x98tyrosine-containingxe2x80x99 cyclophilins in a method for identifying compounds capable of binding to and/or inhibiting the enzymatic activity of these proteins. Such compounds may be further screened for their ability to adversely affect parasites which are not susceptible to the anti-parasitic effects of CsA.
Generally, this method comprises contacting a xe2x80x98tyrosine-containingxe2x80x99 cyclophilin with a compound to be tested (test compound) and measuring, binding and/or the change in enzymatic activity. Such methods may include the following general methods for drug discovery, familiar to the skilled artisan, but not limited to, using the tyrosine-containing cyclophilin to screen natural products (Hazuda, et al., Drug Des. Discov., 15:17-24 (1997)), any type of combinatorial library of synthetic molecules (Hazuda, et al., Drug Des. Discov., supra, Lam, Anticancer Drug Des., 12:145-167 (1997)), phage display libraries (Fang, et al., Biochem. Biophys. Res. Commun., 220:53-56 (1996)). Because of the high degree of homology between cyclophilins, preferably, the test compound is a CsA derivative. Most preferably, the CsA derivative is a binding site derivative. In particular, this method can be used (i) to screen for CsA derivatives capable of binding to filarial xe2x80x98tyrosine-containingxe2x80x99 cyclophilins that inhibit PPlase activity and (ii) to select those which are less or non-immunosuppressive to the host.
In a preferred embodiment, a fusion protein comprising the xe2x80x98tyrosine-containingxe2x80x99 cyclophilin and protein having binding affinity for a substrate, e.g., malE, is used in an affinity chromatography system to screen and select binding compounds. In this method, the fusion protein is contacted with a substrate to which the binding protein has specific affinity such that the fusion protein is reversibly affixed to the column. A test compound is then added to the column. The compound may be labeled. The column is then washed and analyzed to determine the presence of the compounds. Compounds found to have binding affinity for the fusion protein can then be tested for the ability to inhibit PPlase activity.
One xe2x80x98tyrosine-containingxe2x80x99 cyclophilin useful in the method of the present invention is from a parasitic nematode, Dirofilaria immitis, the causative agent of heartworm disease in dogs and cats. Others include the xe2x80x98tyrosine-containingxe2x80x99 cyclophilin from the human parasites Onchocerca volvulus and Brugia malayi. 
The present invention further relates to the use the DNA encoding the D.immitis xe2x80x98tyrosine-containingxe2x80x99 cyclophilin, or a fragment thereof, in the identification and isolation of related genes from other organisms, including other species of parasitic nematodes. The present inventors have determined the presence of related genes by PCR in B.malayi and O.volvulus. These genes will hybridize to the D. immitis xe2x80x98tyrosine-containingxe2x80x99 cyclophilin under suitable conditions described in the Examples below.